title: Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h.
abstract: The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne's disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne's disease.
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK. stxbs@nottingham.ac.uk
authors.forename: Benjamin M C
authors.initials: BM
authors.lastname: Swift
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Denton
authors.forename: Sophie A
authors.initials: SA
authors.lastname: Mahendran
authors.forename: Jonathan N
authors.initials: JN
authors.lastname: Huxley
authors.forename: Catherine E D
authors.initials: CE
authors.lastname: Rees
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D001435:Bacteriophages / Q000235:genetics*; D002417:Cattle; D004269:DNA, Bacterial / Q000032:analysis / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D057230:Limit of Detection; D059346:Magnets; D008892:Milk / Q000382:microbiology; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000302:isolation & purification; D010283:Paratuberculosis / Q000175:diagnosis / Q000382:microbiology; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012756:Sheep
chemical_list: D004269:DNA, Bacterial
keywords: Bacteriophage; FASTplaqueTB assay; FPTB; Johne's disease; MP; Magnetic separation; Media Plus; PMMS; Paratuberculosis; Peptide mediated magnetic separation; Rapid detection